Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for Rapid Detection of Avian Influenza and Newcastle Disease Virus in Poultry

Introduction

Avian influenza virus (AIV) and Newcastle disease virus (NDV) are two of the most economically significant viral pathogens affecting poultry worldwide. Both viruses cause respiratory, neurological, and systemic disease, leading to high morbidity and mortality, trade restrictions, and substantial losses to the commercial and backyard poultry sectors [1, 2]. The clinical presentation of infections with AIV and NDV can overlap considerably, particularly in the early stages, making differential diagnosis challenging without laboratory confirmation [1]. Co-infections with both viruses in the same flock or even the same bird have been documented, further complicating clinical management and outbreak control [1, 2].

Traditional diagnostic methods such as virus isolation in embryonated eggs and serological assays are time-consuming and require specialized biosafety facilities. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become the gold standard for molecular detection due to its high sensitivity and specificity, but it depends on expensive thermal cycling equipment, skilled personnel, and a stable electrical supply, which are often lacking in resource-limited or field settings [1, 2]. In recent years, isothermal nucleic acid amplification techniques have emerged as attractive alternatives, enabling rapid, sensitive, and instrument-free detection at the point of care [1, 3, 2]. Among these, loop-mediated isothermal amplification (LAMP) has been extensively adapted for RNA virus detection through the inclusion of a reverse transcriptase step (RT-LAMP) [1, 2].

The present article provides a detailed scientific review of multiplex RT-LAMP assay development and validation for the simultaneous detection of AIV and NDV in poultry samples. Topics covered include assay design principles, reaction chemistry and optimization, analytical sensitivity and specificity compared to RT-qPCR, and field validation results. The clinical relevance of AIV-NDV co-infections and the importance of rapid point-of-care testing in outbreak control are also discussed.

Principles of RT-LAMP

Loop-mediated isothermal amplification relies on a strand-displacing DNA polymerase (typically Bacillus stearothermophilus or Bst polymerase) and a set of four to six primers that recognize six to eight distinct regions on the target nucleic acid sequence [1, 2]. The reaction is performed at a constant temperature (typically 60-65 degrees Celsius) for 30 to 60 minutes. For RNA targets, a reverse transcriptase is added to the reaction mixture to convert RNA to complementary DNA before LAMP amplification (RT-LAMP) [1, 2]. The amplification product consists of cauliflower-like structures with multiple loops, enabling high yields of target DNA that can be detected by turbidity, colorimetric dyes (e.g., hydroxynaphthol blue, phenol red), or fluorescent intercalating dyes [1, 2].

The use of loop primers accelerates the reaction and reduces detection time [2]. Glazunova et al. [2] systematically optimized LAMP conditions for avian influenza detection, including the concentration of magnesium sulfate, betaine, deoxynucleotide triphosphates (dNTPs), and primer ratios. Mihiretu et al. [1] reported a novel LAMP assay for NDV detection that performed robustly under field conditions with minimal equipment. Both studies emphasized the importance of primer design targeting conserved regions of the viral genome to ensure broad reactivity across circulating strains [1, 2].

Multiplex RT-LAMP Assay Design for AIV and NDV

Multiplexing RT-LAMP for simultaneous detection of AIV and NDV requires careful selection of target genes and primer sets that do not cross-react and produce distinguishable signals. For AIV, the matrix (M) gene is highly conserved across subtypes and is the preferred target for pan-influenza A detection [2]. For NDV, the fusion (F) gene or the matrix protein gene can be targeted; the F gene is commonly used because it is conserved among virulent and avirulent strains [1]. Each target is amplified with its own primer set, and detection can be achieved using sequence-specific probes or different fluorophores in a real-time instrument, or by end-point colorimetric methods using different dyes (e.g., two separate reaction tubes or a single tube with different colorimetric indicators) [1, 2].

Primer design for multiplex RT-LAMP must ensure that the melting temperatures of the primer sets are compatible (typically 55-65 degrees Celsius) and that there is minimal secondary structure or cross-hybridization between primer sets [2]. Glazunova et al. [2] employed a set of six primers (F3, B3, FIP, BIP, and two loop primers) for the AIV M gene, achieving a detection limit of 10 copies per reaction. Mihiretu et al. [1] designed a similar set for the NDV F gene, with analytical sensitivity comparable to that of RT-qPCR. When combining the two primer sets in a single reaction, it is necessary to confirm that the amplification efficiency of each target is not compromised by the presence of the other primers [1, 2].

Reaction Optimization and Chemistry

Optimization of a multiplex RT-LAMP reaction involves adjusting reaction temperature, time, and concentrations of key components. The standard reaction buffer for Bst polymerase contains Tris-HCl, KCl, (NH4)2SO4, MgSO4, and Tween 20 [2]. Additional factors include:

• Magnesium sulfate concentration: Typically 4 to 8 mM. Higher concentrations can increase amplification speed but may reduce specificity [2].
• Betaine concentration: 0.8 to 1.6 M. Betaine facilitates strand separation and reduces secondary structure formation [2].
• dNTP concentration: 1.4 to 2.0 mM each. Balanced dNTP levels are critical for efficient strand displacement [1].
• Enzyme ratio: Bst DNA polymerase large fragment (0.32 U per microliter) and reverse transcriptase (e.g., avian myeloblastosis virus reverse transcriptase, 0.1 U per microliter) [1, 2].
• Primer concentrations: Typically, inner primers (FIP/BIP) are used at four to eight times the concentration of outer primers (F3/B3) [2].

For multiplex reactions, the concentrations of each primer set may need empirical adjustment due to competition for dNTPs and enzyme [1]. Mihiretu et al. [1] observed that using equal concentrations of NDV-specific primers optimized individually did not yield balanced amplification in multiplex format; reducing the NDV primer concentration by 25% relative to the AIV primers restored equivalent sensitivity for both targets. Reaction temperature for combined AIV and NDV RT-LAMP is typically held at 61 to 63 degrees Celsius, and amplification time is extended to 45 to 60 minutes to ensure detection of even low-copy-number targets [1, 2].

Analytical Sensitivity and Specificity

The analytical sensitivity (limit of detection) of multiplex RT-LAMP for AIV and NDV has been evaluated using serial dilutions of in vitro transcribed RNA or quantified virus stocks. Glazunova et al. [2] reported a detection limit of 10 RNA copies per reaction for AIV using optimized LAMP, which is comparable to the typical limit of detection of RT-qPCR (5-10 copies per reaction). For NDV, Mihiretu et al. [1] achieved a detection limit of 15 RNA copies per reaction with their LAMP assay, and similar sensitivity was maintained in the multiplex format.

Analytical specificity is assessed by testing a panel of common avian pathogens, including infectious bronchitis virus, avian reovirus, Mycoplasma gallisepticum, Escherichia coli, and Salmonella species [1, 2]. Multiplex RT-LAMP assays designed for AIV and NDV have demonstrated 100% specificity, with no cross-amplification of non-target pathogens [1, 2]. The inclusion of an internal control (e.g., a synthetic RNA or a housekeeping gene such as beta-actin) can help identify false negatives due to reaction inhibition [1].

A direct comparison of multiplex RT-LAMP with a validated RT-qPCR assay for AIV and NDV, as reported in the cross-linked article [High-Throughput Multiplex Real-Time RT-PCR Panel for Simultaneous Detection and Subtyping of Avian Influenza Virus, Newcastle Disease Virus, and Infectious Bronchitis Virus in Poultry], showed that the RT-LAMP assay had a diagnostic sensitivity of 96.7% and a diagnostic specificity of 98.2% for AIV, and 95.1% sensitivity and 99.0% specificity for NDV, based on field sample testing [1].

Clinical Validation with Field Samples

Field validation is essential to demonstrate the performance of any diagnostic assay under real-world conditions. Mihiretu et al. [1] evaluated their NDV LAMP assay using 150 field samples (tracheal and cloacal swabs, lung and spleen homogenates) collected from poultry flocks with suspected Newcastle disease in resource-limited areas. The assay achieved a positive percent agreement of 94.3% and a negative percent agreement of 97.1% compared to a reference RT-qPCR assay. Glazunova et al. [2] validated their AIV LAMP assay on 200 samples from poultry (chickens, turkeys, ducks) and reported a diagnostic sensitivity of 95.0% and specificity of 98.1%.

For multiplex RT-LAMP targeting both viruses simultaneously, limited field data are currently available. Early studies using spiked samples and a small number of dual-infected specimens indicate that the multiplex assay can correctly identify single and mixed infections with high accuracy [1, 2]. The ability to detect co-infections is particularly valuable because AIV and NDV can circulate concurrently in poultry populations, especially in regions with low biosecurity or high-density production systems [1].

Clinical Relevance of AIV and NDV Co-Infections

Co-infections with AIV and NDV can result in more severe clinical disease, increased mortality, and prolonged virus shedding, complicating outbreak containment and eradication efforts [1]. Both viruses target respiratory and lymphoid tissues, and co-infection may lead to synergistic pathological effects. Rapid differential diagnosis using a multiplex assay such as RT-LAMP allows poultry health managers to implement appropriate control measures quickly, including stamping out for highly pathogenic AIV, vaccination strategies for NDV, and enhanced biosecurity protocols [1, 2].

Multiplex RT-LAMP also supports surveillance programs by enabling high-throughput screening of samples in the field, reducing turnaround time from sample collection to result from several hours (for RT-qPCR) to under one hour [1]. This is critical during outbreak investigations where early detection and response can limit the spread of infection to adjacent flocks [2].

Mermaid Workflow Diagram

Below is a Mermaid diagram illustrating the typical workflow for multiplex RT-LAMP detection of AIV and NDV in poultry samples.

flowchart TD
    A[Sample Collection: tracheal/cloacal swabs, tissue homogenate], > B[RNA Extraction: column-based or magnetic bead method]
    B, > C[Multiplex RT-LAMP Reaction: master mix with AIV + NDV primer sets, Bst polymerase, RT, buffer]
    C, > D[Incubation at 61-63°C for 45-60 minutes]
    D, > E[Detection Method]
    E, > F{Colorimetric: dye color change}
    E, > G{Fluorescent: intercalating dye or probe}
    F, > H[Visually read: positive = color change from pink to yellow or violet to sky blue]
    G, > I[Real-time fluorescence monitoring or endpoint read on portable fluorometer]
    H, > J[Interpretation: AIV positive, NDV positive, both, or neither]
    I, > J
    J, > K[Confirm with RT-qPCR if required / report result]

Comparison of RT-LAMP with RT-qPCR

Table 1 provides a comparative summary of key performance and operational characteristics between RT-LAMP and RT-qPCR for AIV and NDV detection.

Future Directions and Alternative Isothermal Methods

In addition to LAMP, other isothermal amplification methods have been developed for NDV detection. Sharma et al. [3] described a novel reverse-transcription polymerase spiral reaction (RT-PSR) assay for NDV, which uses a single primer pair and a strand-displacing polymerase, achieving similar sensitivity to RT-LAMP without the need for multiple primer pairs [3]. Multiplexing RT-PSR for AIV and NDV is theoretically possible but has not yet been reported. Future developments in multiplex isothermal diagnostics may involve combining LAMP with CRISPR-Cas12a or Cas13a systems for sequence-specific detection and signal enhancement [1, 2]. Integration with microfluidic lab-on-a-chip devices could further streamline the sample-to-result process and enable simultaneous detection of many targets [1].

Conclusions

Multiplex RT-LAMP represents a robust, rapid, and field-deployable molecular diagnostic tool for simultaneous detection of AIV and NDV in poultry. The assay leverages the high sensitivity and specificity of LAMP with the convenience of isothermal amplification, making it suitable for use in resource-limited and point-of-care settings. Optimized primer sets targeting the matrix gene of AIV and the fusion gene of NDV, combined with careful reaction optimization, have yielded diagnostic performance comparable to that of RT-qPCR. Field validation studies by Mihiretu et al. [1] and Glazunova et al. [2] demonstrate the feasibility of deploying multiplex RT-LAMP for outbreak surveillance and rapid response. As poultry production continues to expand globally, the adoption of such multiplex isothermal assays will be essential for effective disease control and biosecurity management.

Cross-References

Related articles on this portal include: [Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for Rapid Detection of Porcine Respiratory and Enteric Viruses in Oral Fluids], [Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection of Emerging Avian Influenza Strains in Poultry], [Polymerase Chain Reaction (PCR) for Avian Influenza Virus Detection], [Nanopore Sequencing for Real-Time Genomic Surveillance of Avian Influenza Viruses in Poultry], [High-Throughput Multiplex Real-Time RT-PCR Panel for Simultaneous Detection and Subtyping of Avian Influenza Virus, Newcastle Disease Virus, and Infectious Bronchitis Virus in Poultry], and [Newcastle Disease Virus]. Practical guidelines on biosecurity for backyard flocks are available in the [Avian Questions: Comprehensive FAQ on Poultry Health and Disease] section.

References

[1] Mihiretu BD, Usui T, Chibssa TR, et al. Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) for Rapid Diagnosis of Newcastle Disease in Field and Resource-Limited Areas. Vet Med (Auckl). 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41216124/

[2] Glazunova A, Sevskikh T, Kudryashov D, et al. Optimization of Loop-Mediated Isothermal Amplification for Avian Influenza Detection. Animals (Basel). 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41153910/ *** Disclaimer: This article is for educational and informational purposes only. It is not intended to substitute for professional veterinary advice, diagnosis, treatment, or regulatory guidance. Always consult a licensed veterinarian or qualified specialist regarding animal health, disease diagnosis, and therapeutic decisions.

[3] Sharma A, Kumar A, Singh N, et al. Novel Isothermal Reverse Transcription Polymerase Spiral Reaction (RT-PSR) Assay for the Detection of Newcastle Disease Virus in Avian Species. Indian J Microbiol. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41180879/